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Movement of voltage sensor S4 in domain 4 is tightly coupled to sodium channel fast inactivation and gating charge immobilization.

Kühn FJ, Greeff NG - J. Gen. Physiol. (1999)

Bottom Line: The double mutant R1635/1638H shows intermediate effects on inactivation.Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H.These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state.

View Article: PubMed Central - PubMed

Affiliation: Physiologisches Institut, Universität Zürich, CH-8057 Zürich, Switzerland.

ABSTRACT
The highly charged transmembrane segments in each of the four homologous domains (S4D1-S4D4) represent the principal voltage sensors for sodium channel gating. Hitherto, the existence of a functional specialization of the four voltage sensors with regard to the control of the different gating modes, i.e., activation, deactivation, and inactivation, is problematic, most likely due to a functional coupling between the different domains. However, recent experimental data indicate that the voltage sensor in domain 4 (S4D4) plays a unique role in sodium channel fast inactivation. The correlation of fast inactivation and the movement of the S4D4 voltage sensor in rat brain IIA sodium channels was examined by site-directed mutagenesis of the central arginine residues to histidine and by analysis of both ionic and gating currents using a high expression system in Xenopus oocytes and an optimized two-electrode voltage clamp. Mutation R1635H shifts the steady state inactivation to more hyperpolarizing potentials and drastically increases the recovery time constant, thereby indicating a stabilized inactivated state. In contrast, R1638H shifts the steady state inactivation to more depolarizing potentials and strongly increases the inactivation time constant, thereby suggesting a preferred open state occupancy. The double mutant R1635/1638H shows intermediate effects on inactivation. In contrast, the activation kinetics are not significantly influenced by any of the mutations. Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H. The time courses of recovery from inactivation and immobilization correlate well in wild-type and mutant channels, suggesting an intimate coupling of these two processes that is maintained in the mutations. These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state. Moreover, the presented data strongly suggest that S4D4 is involved in the control of fast inactivation.

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Scheme of possible interactions of the central arginines of S4D4 with a putative counter-charge that could explain the different inactivation behavior of the mutants. Illustrated is the central section of S4D4 containing the highly conserved central arginines R3–R6; the hypothetical negative counter-charge is indicated. WT and R5H are shown in the open state and R4H and R4/5H are shown in the inactivated state. Compared with WT, the O → I transition is considerably slowed in R5H (indicated by a thin arrow) and moderately slowed in R4H (not illustrated). This is due to the electrostatic asymmetry resulting from the interaction of the negative counter-charge with the neighbored arginines and histidines in the single mutants. On the other hand, the I2 → C2 transition (recovery from inactivation) is for the same reason drastically slowed in R4H (indicated by a thin arrow) and hardly affected in R5H (not illustrated). In contrast, WT and R4/5H display more symmetrical electrostatics, but due to the considerably altered structure in the double mutant, the mobility of S4D4 is slowed in both directions.
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Figure 9: Scheme of possible interactions of the central arginines of S4D4 with a putative counter-charge that could explain the different inactivation behavior of the mutants. Illustrated is the central section of S4D4 containing the highly conserved central arginines R3–R6; the hypothetical negative counter-charge is indicated. WT and R5H are shown in the open state and R4H and R4/5H are shown in the inactivated state. Compared with WT, the O → I transition is considerably slowed in R5H (indicated by a thin arrow) and moderately slowed in R4H (not illustrated). This is due to the electrostatic asymmetry resulting from the interaction of the negative counter-charge with the neighbored arginines and histidines in the single mutants. On the other hand, the I2 → C2 transition (recovery from inactivation) is for the same reason drastically slowed in R4H (indicated by a thin arrow) and hardly affected in R5H (not illustrated). In contrast, WT and R4/5H display more symmetrical electrostatics, but due to the considerably altered structure in the double mutant, the mobility of S4D4 is slowed in both directions.

Mentions: Accordingly, it is conceivable that R4 and R5 could be critical determinants for the voltage-driven shift of R2 and R3 involved in the structural interactions that are necessary for this movement. With respect to the observed antagonism, we propose that negative counter charges affect the movement of the positively charged S4D4 residues inside the hydrophobic protein core. For Shaker potassium channels it was demonstrated that there are electrostatic interactions between the positively charged residues of the central to innermost section in S4 and the negatively charged residues in S2 (Papazian et al. 1995). Histidine has, depending on the local protein environment, a pK of 5.6–7.0 and therefore should carry less positive charge than arginine at physiological pH (Sancho et al. 1992). However, this means that the mutation of arginine to histidine most likely is not completely charge neutralizing. It is obvious that the side chains of histidine and arginine have a distinctly different molecular structure. This gives rise to the assumption that the mutations R4H, R4/5H, and R5H could markedly change the local structure and at least partially change the charge distribution (Fig. 9).


Movement of voltage sensor S4 in domain 4 is tightly coupled to sodium channel fast inactivation and gating charge immobilization.

Kühn FJ, Greeff NG - J. Gen. Physiol. (1999)

Scheme of possible interactions of the central arginines of S4D4 with a putative counter-charge that could explain the different inactivation behavior of the mutants. Illustrated is the central section of S4D4 containing the highly conserved central arginines R3–R6; the hypothetical negative counter-charge is indicated. WT and R5H are shown in the open state and R4H and R4/5H are shown in the inactivated state. Compared with WT, the O → I transition is considerably slowed in R5H (indicated by a thin arrow) and moderately slowed in R4H (not illustrated). This is due to the electrostatic asymmetry resulting from the interaction of the negative counter-charge with the neighbored arginines and histidines in the single mutants. On the other hand, the I2 → C2 transition (recovery from inactivation) is for the same reason drastically slowed in R4H (indicated by a thin arrow) and hardly affected in R5H (not illustrated). In contrast, WT and R4/5H display more symmetrical electrostatics, but due to the considerably altered structure in the double mutant, the mobility of S4D4 is slowed in both directions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230646&req=5

Figure 9: Scheme of possible interactions of the central arginines of S4D4 with a putative counter-charge that could explain the different inactivation behavior of the mutants. Illustrated is the central section of S4D4 containing the highly conserved central arginines R3–R6; the hypothetical negative counter-charge is indicated. WT and R5H are shown in the open state and R4H and R4/5H are shown in the inactivated state. Compared with WT, the O → I transition is considerably slowed in R5H (indicated by a thin arrow) and moderately slowed in R4H (not illustrated). This is due to the electrostatic asymmetry resulting from the interaction of the negative counter-charge with the neighbored arginines and histidines in the single mutants. On the other hand, the I2 → C2 transition (recovery from inactivation) is for the same reason drastically slowed in R4H (indicated by a thin arrow) and hardly affected in R5H (not illustrated). In contrast, WT and R4/5H display more symmetrical electrostatics, but due to the considerably altered structure in the double mutant, the mobility of S4D4 is slowed in both directions.
Mentions: Accordingly, it is conceivable that R4 and R5 could be critical determinants for the voltage-driven shift of R2 and R3 involved in the structural interactions that are necessary for this movement. With respect to the observed antagonism, we propose that negative counter charges affect the movement of the positively charged S4D4 residues inside the hydrophobic protein core. For Shaker potassium channels it was demonstrated that there are electrostatic interactions between the positively charged residues of the central to innermost section in S4 and the negatively charged residues in S2 (Papazian et al. 1995). Histidine has, depending on the local protein environment, a pK of 5.6–7.0 and therefore should carry less positive charge than arginine at physiological pH (Sancho et al. 1992). However, this means that the mutation of arginine to histidine most likely is not completely charge neutralizing. It is obvious that the side chains of histidine and arginine have a distinctly different molecular structure. This gives rise to the assumption that the mutations R4H, R4/5H, and R5H could markedly change the local structure and at least partially change the charge distribution (Fig. 9).

Bottom Line: The double mutant R1635/1638H shows intermediate effects on inactivation.Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H.These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state.

View Article: PubMed Central - PubMed

Affiliation: Physiologisches Institut, Universität Zürich, CH-8057 Zürich, Switzerland.

ABSTRACT
The highly charged transmembrane segments in each of the four homologous domains (S4D1-S4D4) represent the principal voltage sensors for sodium channel gating. Hitherto, the existence of a functional specialization of the four voltage sensors with regard to the control of the different gating modes, i.e., activation, deactivation, and inactivation, is problematic, most likely due to a functional coupling between the different domains. However, recent experimental data indicate that the voltage sensor in domain 4 (S4D4) plays a unique role in sodium channel fast inactivation. The correlation of fast inactivation and the movement of the S4D4 voltage sensor in rat brain IIA sodium channels was examined by site-directed mutagenesis of the central arginine residues to histidine and by analysis of both ionic and gating currents using a high expression system in Xenopus oocytes and an optimized two-electrode voltage clamp. Mutation R1635H shifts the steady state inactivation to more hyperpolarizing potentials and drastically increases the recovery time constant, thereby indicating a stabilized inactivated state. In contrast, R1638H shifts the steady state inactivation to more depolarizing potentials and strongly increases the inactivation time constant, thereby suggesting a preferred open state occupancy. The double mutant R1635/1638H shows intermediate effects on inactivation. In contrast, the activation kinetics are not significantly influenced by any of the mutations. Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H. The time courses of recovery from inactivation and immobilization correlate well in wild-type and mutant channels, suggesting an intimate coupling of these two processes that is maintained in the mutations. These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state. Moreover, the presented data strongly suggest that S4D4 is involved in the control of fast inactivation.

Show MeSH